Elevated Circulating Tumor Cells Research Articles

Abstract A003: Characterization of circulating tumor cells from patients with metastatic bladder cancer

Abstract Assessing the molecular profile of bladder tumors from patients with metastatic disease is crucial for implementing personalized treatment. Current standards are based on highly invasive biopsies. Alternatively, liquid biopsies of tumor cells, proteins, and nucleic acids allows longitudinal profiling in a less invasive manner. Circulating tumor cells (CTCs) isolated from blood are a key analyte of liquid biopsy. In this study, we developed a protein and transcriptomic co-analysis workflow for bladder cancer CTCs utilizing the graphene oxide (GO) microfluidic chip conjugated with antibodies against both EpCAM and EGFR to isolate CTCs from blood of patients with metastatic bladder cancer. Following CTC capture, protein and mRNA were analyzed using immunofluorescent staining and ion-torrent whole transcriptome sequencing. This is the first time that whole transcriptome sequencing was used in a downstream application for the GO microfluidic platform. We found that elevated CTC counts were significantly associated with patient disease status at the time of blood draw and that greater than 2 CTCs per mL were associated with shorter overall survival. Invasive markers, including EGFR, HER-2, CD31, and ADAM15, were detected in subpopulations of CTCs. Targeted whole transcriptome sequencing showed distinct expression profiles from patients with metastatic burden at the time of blood draw. In these patients, we found significant upregulation of several metastasis-related and chemotherapy-resistant genes demonstrating the capability of identifying tumor-related RNA signatures from circulating CTCs using immune-affinity GO chip-based assays. In addition, this workflow highlights the prognostic potential of CTCs in metastatic bladder cancer patients. Citation Format: Zeqi Niu, Molly Kozminski, Kathleen C. Day, Luke Broses, Marian L. Henderson, Sophia Merajver, Aaron Udager, Phillip L. Palmbos, Sunitha Nagrath, Mark L. Day. Characterization of circulating tumor cells from patients with metastatic bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A003.

Abstract PO1-16-02: Correlation of Circulating Tumor Cells (CTC) with Clinical Characteristics, Pathological Factors, and Treatment Response in Patients with Metastatic Breast Cancer (MBC)

Abstract Background: Circulating Tumor Cells (CTC) can be isolated in 40-80% of patients with metastatic breast cancer (MBC). High levels of CTC, defined as ≥5/7.5 ml of blood are associated with shorter progression-free survival and overall survival. CTC can be tested for clinically relevant biomarkers, such as ER, and HER2 status, and have the potential to guide therapy as well as help with disease monitoring. We investigated CTC detection rate, its correlation with clinical characteristics, pathological characteristics, and response to treatment in patients with any type of metastatic breast cancer who progressed on at least one line of therapy. Methods: Eligible patients with MBC were enrolled in the IMAGE-II study (Individualized Molecular Analyses Guide Efforts in Breast Cancer) NCT02965755, in which we obtained archival tissue as part of the standard of care, and prospectively collected serial blood samples for CTC analysis. We compared CTC levels and biomarker status with clinical factors, tissue-based pathology, and molecular biomarkers. We assessed whether changes in CTC count correlated with treatment response. Blood samples were collected at baseline (Day 1), after 1-2 weeks of therapy, after 8-12 weeks, and at subsequent restaging (every 8-12 weeks). Medical records were reviewed every 3 months for ongoing treatment response, and death. Samples were processed at the Biocept CLIA-certified Laboratory. CTC were enumerated by the presence of CD45- and DAPI+ cells. Further CTC characterization was performed by antibody staining of specific protein biomarkers (ER, HER2). Fisher’s exact test was used to evaluate the association between baseline CTC < 5 and ≥5/7.5ml with patient characteristics. McNemar’s test was used to assess discordance between tissue-based and CTC-defined markers. Results: Between 1/26/2018 and 12/31/22 baseline samples were collected from 70 women with a median age of 56 (36-82) who were enrolled at four study sites. CTC was detected in 59 (84%) of participants. Baseline CTC counts were < 5/7.5 ml in 37/59 (63%) and ≥5/7.5 ml in 22/59 (37%) of participants. Differences between breast cancer subtype and CTC-receptor status were observed (Table 1). Among the 41 patients who were ER-positive, 31 (76%) were CTC ER-negative(p< 0.001). Among 29 patients who were ER-negative, 4 (14%) were CTC ER-Positive (P< 0.001). Among the 7 patients who were HER2 positive, 2(29%) were HER2 positive on CTC. Among 63 patients who were HER2 negative, 7(11%) had HER2 expression on their CTC. The discordance in the classification of HER2 wasn't statistically significant (p=0.77). Elevated CTC at baseline was more frequently detected in younger participants (< 50 years old) (55% vs 27%; P=0.22), in Black women compared to White (60 % vs 29 %, P=0.27), and in participants with visceral vs non-visceral metastasis (52% vs 28 %, P=0.40). Patients with CTC ≥5 vs < 5/7.5mL at baseline had a shorter duration of anti-HER2-based therapy (61.5 vs 836 days, p=0.04). There were no statistically significant differences among participants who received chemotherapy agents (316.5 vs 365.5 days, P=0.88), endocrine therapy (376 vs 490 days, P=0.89), or overall therapy (272 vs 390 days, P=0.21). Conclusions: We observed significant differences in the expression of ER between tumor tissue and CTC, which can be partly due to tumor evolution over time. Additionally, participants who are young, Black, and those who have visceral metastasis may have higher CTC counts. Higher CTC counts were associated with a shorter duration of anti-HER2 therapy. Although it didn’t meet statistical significance, a similar trend was observed in patients who received chemotherapy and endocrine therapy. Table 1: MBC Tissue-Based Subtype and CTC-receptor status at baseline Citation Format: Asnakech Bayable, Tingchang Wang, Amanda Blackford, Jessica Tao, Jenna Canzoniero, Seoho Lee, Faith Too, Barbara Blouw, Mary Wilkinson, Rima Couzi, Antonio Wolff, Christie Hiton, Ben Park, Vered Stearns, Cesar Santa-Maria. Correlation of Circulating Tumor Cells (CTC) with Clinical Characteristics, Pathological Factors, and Treatment Response in Patients with Metastatic Breast Cancer (MBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-02.

Abstract PO5-16-05: Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer, comprising only between 1-5% of all breast cancer cases in the United States. Despite its low incidence, IBC accounts for 10% of breast cancer-related deaths and is often diagnosed with metastasis, resulting in shorter survival compared to non-IBC cases. The 5-year relative survival rate for all stages of IBC is 39%, dropping to 19% for patients with distant metastasis. Liquid biopsy-based biomarkers have emerged as potential analytes for predicting prognosis and monitoring response to therapies in IBC patients. We explored the prognostic role of liquid biopsy encompassing both CTCs and cfDNA in the blood of patients with stage III/IV IBC and their clinical outcomes. Methods: CTC and cfDNA analysis was performed on 90 blood samples from 47 patients with stage III and IV IBC. Samples were collected before (pre-treatment, n=43) initiation and after (post-treatment, n=47) completion of neoadjuvant chemotherapy. CTCs were enriched and enumerated using the CellSearch® platform (Menarini-Silicon Biosystems). CfDNA was extracted from plasma using MagMax™ Cell-Free Total Nucleic Acid Isolation Kit (Applied Biosystems™) and quantified using the Qubit™ dsDNA High Sensitivity kit (Invitrogen™). Differences in cfDNA concentrations were used to plot a receiver operating characteristic (ROC) curve which was then used to determine Area Under the Curve (AUC) and a cfDNA cut-off point. ROC cut-off was calculated based on Youden Index J. Survival analysis was performed by the Kaplan-Meier estimator and Cox regression models. P-values were measured using the Student t-test. Results: Prior to neoadjuvant treatment, CTC was detected in 51% of patient samples, and the median cfDNA concentration was 5.87 ng/mL (range 0.95-24.11 ng/mL). After completion of neoadjuvant treatment, CTC was detected in 25% of patient samples, and the median cfDNA concentration was 11.19 ng/mL (range 3.41-88.13 ng/mL). The cut-off points determined for pre-treatment and post-treatment cfDNA concentration was >4.2 ng/mL and >9.5 ng/mL respectively. At pre-treatment, CTC detection and cfDNA concentration >4.2 ng/mL were independently associated with disease relapse (P=0.007, 0.016, respectively). The combination of CTC detection and cfDNA concentration >4.2 ng/mL was significantly associated with disease relapse (P=0.0004), decreased relapse free survival (RFS, P=0.005) and overall survival (OS, P=0.007). At the post-neoadjuvant treatment time point, the combination of CTC detection and cfDNA concentration >9.5 ng/mL was significantly associated with relapse regardless of pCR status (P=0.02). Conclusion: The combined presence of CTCs and elevated cfDNA concentrations in patients with stage III/IV IBC after completion of neoadjuvant chemotherapy was associated with disease relapse regardless of pCR status. A combination of elevated cfDNA and CTC detection signatures after completion of neoadjuvant chemotherapy may be useful for risk stratification and adjuvant systemic therapy planning. Citation Format: Sridevi Addanki, Salyna Meas, Vanessa Sarli, Abdulkader Almosa, Wendy Woodward, Dhruvajyoti Roy, Anthony Lucci. Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-05.

Circulating tumor cells in patients with hormone receptor-positive, HER2-negative metastatic breast cancer receiving palbociclib with fulvestrant after progression on palbociclib with an aromatase inhibitor.

e13018 Background: High levels of circulating tumor cells (CTCs) at initiation of therapy in patients with metastatic breast cancer (MBC) are associated with worse prognosis. In patients with hormone receptor–positive (HR+), HER2-negative MBC, the benefit of maintaining CDK4/6 inhibitor (CDK4/6i) after progression on first-line therapy with endocrine therapy and CDK4/6i is not fully defined. To study the relationship between CTCs, tumor characteristics, and clinical outcomes, we enumerated CTC dynamics in patients with HR+ HER2- MBC enrolled on a phase II, multi-center, open-label interventional trial of palbociclib and fulvestrant after disease progression on prior palbociclib with an aromatase inhibitor (NCT02738866). Methods: Blood samples from study participants were collected at baseline (pretreatment), cycle 2 (C2, 8 weeks after initiation of fulvestrant and palbociclib), and at disease progression. Using a previously analytically validated assay (CellSearch), CTCs were enumerated. Progression-free survival (PFS) was estimated using the Kaplan Meier method. The relationship between CTC count at baseline (< 5, >5/7.5 ml Whole Blood (WB)) and PFS was analyzed using Cox proportional hazards regression. The association between PFS and CTC count among patients with baseline CTC ≥5 was estimated with a Cox proportional hazards model, adjusting for age, race, and progesterone receptor (PgR). Results: Between May 2017 and September 2022, baseline samples were collected from 52 of 60 trial participants enrolled at 5 centers in the United States, mean age 55 years (SD 14). Baseline CTC counts were <5 in 30/52 (58%) and >5 in 22/52 (42%) of participants. Among participants at baseline, only tissue PgR negativity was associated with higher likelihood of having elevated CTCs >5 (p = 0.04). Association of CTCs >5 and select patient and tumor characteristics is shown in Table 1. There was no statistically significant association between continuously measured CTC and PFS among those with elevated CTCs at baseline. Median duration of therapy among the entire 52 participant cohort was 4 months. Median PFS in participants with baseline CTC <5 vs >5/7.5 ml WB was 7.83 (95% CI [5.37, 12.60]) and 2.83 (95% CI [1.77, 4.73]) months, respectively (HR): 2.10, 95% CI [1.15, 3.82], p = 0.01). Conclusions: Baseline CTCs >5/7.5 ml WB conferred a 2-fold increased risk of disease progression. Tissue PgR negativity was associated with baseline CTCs >5. These data can be used to inform new studies in which baseline and early change in CTCs may inform second line treatment decisions in women with HR+, HER2- MBC. [Table: see text]

Circulating tumor cell abundance in head and neck squamous cell carcinoma decreases with successful chemoradiation and cetuximab treatment

Head and neck squamous cell carcinoma (HNSCC) is a common and deadly cancer. Circulating tumor cell (CTC) abundance may a valuable, prognostic biomarker in low- and intermediate-risk patients. However, few technologies have demonstrated success in detecting CTCs in these populations. We prospectively collected longitudinal CTC counts from two cohorts of patients receiving treatments at our institution using a highly sensitive device that purifies CTCs using biomimetic cell rolling and dendrimer-conjugated antibodies. In patients with intermediate risk human papillomavirus (HPV)-positive HNSCC, elevated CTC counts were detected in 13 of 14 subjects at screening with a median of 17 CTC/ml (range 0.2–2986.5). A second cohort of non-metastatic, HPV- HNSCC subjects received cetuximab monotherapy followed by surgical resection. In this cohort, all subjects had elevated baseline CTC counts median of 73 CTC/ml (range 5.4–332.9) with statistically significant declines during treatment. Interestingly, two patients with recurrent disease had elevated CTC counts during and following treatment, which also correlated with growth of size and ki67 expression in the primary tumor. The results suggest that our device may be a valuable tool for evaluating the success of less intensive treatment regimens.

Circulating tumor cell quantification during abiraterone plus prednisone therapy may estimate survival in metastatic castration-resistant prostate cancer patients.

Circulating tumor cells (CTCs) predict survival in response to different interventions in metastatic castration-resistant prostate cancer (mCRPC) patients. This study aimed to explore the dynamic change in CTCs during abiraterone plus prednisone therapy and its optimal threshold for prognostication in mCRPC patients. CTCs in blood samples from mCRPC patients (N = 98) at baseline and in the 2nd month after abiraterone plus prednisone treatment initiation (M2) were enumerated by using the CellSearch System. CTCs were detected in 64.8% of mCRPC patients at baseline with a median value (interquartile range) of 2.0 (0.0-4.0). Elevated CTC count was related to visceral metastasis (P = 0.003), high alkaline phosphatase (P = 0.043), and high lactate dehydrogenase (P = 0.007). Baseline CTC ≥ 1 (vs. < 1) was only associated with shortened radiographic progression-free survival (rPFS) (P = 0.043); additionally, baseline CTC ≥ 5 (vs. < 5) was linked with unfavorable rPFS (P = 0.037) and overall survival (OS) (P = 0.021). Following the therapy, CTCs were reduced at M2 (P < 0.001). Notably, CTC ≥ 1 (vs. < 1) (P = 0.002) and CTC ≥ 5 (vs. < 5) (P < 0.001) at M2 were related to shortened rPFS according to the Kaplan‒Meier curves, and they could independently estimate deteriorative rPFS in the multivariate Cox regression (P = 0.043 and P = 0.027, respectively). Similarly, CTC ≥ 1 (vs. < 1) (P = 0.022) and CTC ≥ 5 (vs. < 5) (P = 0.002) at M2 were related to shortened OS, whereas only CTC ≥ 5 (vs. < 5) could independently predict unfavorable OS (P = 0.017). CTC count ≥ 5 at M2 exhibits excellent prognostic value for abiraterone plus prednisone therapy in mCRPC patients.

Circulating miR-200 Family and CTCs in Metastatic Breast Cancer before, during, and after a New Line of Systemic Treatment.

The extracellular circulating microRNA (miR)-200 regulates epithelial-mesenchymal transition and, thus, plays an essential role in the metastatic cascade and has shown itself to be a promising prognostic and predictive biomarker in metastatic breast cancer (MBC). Expression levels of the plasma miR-200 family were analyzed in relationship to systemic treatment, circulating tumor cells (CTC) count, progression-free survival (PFS), and overall survival (OS). Expression of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, and CTC status (CTC-positive ≥ 5 CTC/7.5 mL) was assessed in 47 patients at baseline (BL), after the first completed cycle of a new line of systemic therapy (1C), and upon the progression of disease (PD). MiR-200a, miR-200b, and miR-141 expression was reduced at 1C compared to BL. Upon PD, all miR-200s were upregulated compared to 1C. At all timepoints, the levels of miR-200s were elevated in CTC-positive versus CTC-negative patients. Further, heightened miR-200s expression and positive CTC status were associated with poorer OS at BL and 1C. In MBC patients, circulating miR-200 family members decreased after one cycle of a new line of systemic therapy, were elevated during PD, and were indicative of CTC status. Notably, increased levels of miR-200s and elevated CTC count correlated with poorer OS and PFS. As such, both are promising biomarkers for optimizing the clinical management of MBC.

Circulating tumor cells as a predictor for poor prognostic factors and overall survival in treatment naïve oral squamous cell carcinoma patients

The aim of this study was to investigate the presence of circulating tumor cells (CTCs) and their correlation with prognostic factors and clinical outcomes in treatment-naive patients with oral squamous cell carcinoma. CTCs were isolated using OncoDiscover technique from presurgically obtained peripheral blood of 152 patients with treatment naïve oral squamous cell carcinoma. Sensitivity analysis was performed by including 40 healthy controls. CTCs cutoff values for clinicopathologic factors were obtained from receiver operating characteristic curves. Multivariate models determined the significance of CTC as independent variables. Kaplan-Meier analysis differentiated in overall survival between CTC values corresponding to the stage. Sensitivity, specificity, and accuracy of CTC detection were 94.32%, 98%, and 95.17%, respectively. Platform differentiated true positives at >3.5 CTCs (P < .00001). CTCs above 20.5 were suggestive of nodal metastasis (P < .0001) with a linear trend for detecting occult metastasis (P=.061). Early and advanced stages could be differentiated by >13.5 CTCs (P < .0001). Elevated CTCs were significantly associated with extranodal extension (>21.45 CTCs, P=.025), perineural invasion (>19.35 CTCs, P=.049), and depth of invasion (>12.5 CTCs, P=.0038). Median survival was reduced by 19 months when CTCs were >13. Preoperative CTC levels demonstrated a strong correlation with adverse clinicopathology factors and suggested its role as a sensitive prognostic marker to predict survival outcome and disease progress.

Abstract P2-02-02: Differences in levels of circulating tumor cells (CTC) and disseminated tumor cells (DTC) in early-stage lobular versus ductal breast cancer

Abstract Background: Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC). ILC has unique features, such as a diffuse growth pattern due to characteristic loss of E cadherin, and a different pattern of disease metastasis compared to IDC. Prior investigators have shown increased numbers of circulating tumor cells (CTCs) in patients with metastatic ILC versus IDC. We explored the distribution of CTCs and disseminated tumor cells (DTCs) in early stage ILC versus IDC. Methods: We performed a secondary data analysis of the TIPPING study, an institutional review board approved study that pre-operatively collected blood and bone marrow samples from 655 treatment-naïve early-stage breast cancer patients. We analyzed data from 284 patients who had CTCs and DTC enumerated by an EPCAM-based method involving immunomagnetic enrichment and flow cytometry (IE/FC) (61 patients with ILC and 223 patients with IDC). We compared CTC and DTC counts by histology using the Welch Two Sample t-test, linear regression models, as well as ANOVA tests. Multivariate Cox regression analyses were performed to assess association between levels of CTCs/DTCs and clinical outcomes (distant recurrence-free survival [DRFS] and breast cancer-specific survival [BCSS]). Results: ILC tumors were lower grade than IDCs and had a higher proportion of HR+HER2- subtypes (92.00% vs. 75.30%; p&amp;lt;0.001). ILC patients had significantly higher CTC counts than IDC patients (mean 2.11 vs. 0.71 CTCs/mL; p&amp;lt;0.001), a difference that retained significance after adjusting for clinical variables (p=0.003). Additionally, we identified a subset of ILC patients (n = 9; 14.75%) that have elevated CTCs, which was absent in the IDC subset. ILC patients with elevated CTC levels showed no statistically significant association between CTC as a continuous variable with nodal status and breast cancer stage (p=0.26, p=0.25, respectively). In our study, the overall median follow-up was 7.26 years for DRFS and 8.9 years for BCSS. In the subset of ILC patients with elevated CTCs, CTC level as a continuous variable did not show significant association with DRFS or BCSS in a multivariate model adjusting for clinical variables. In the IDC subset, CTC level as a continuous variable did not show significant association with DRFS or BCSS in a multivariate model adjusting for clinical variables.Furthermore, there was no difference in the number of DTCs in ILC versus IDC. DTC level as a continuous variable did not show significant association with DRFS or BCSS in a multivariate model adjusting for clinical variables in both ILC and IDC subsets. Conclusions: Early-stage ILC patients have significantly higher CTC levels than those observed in IDC patients, and we hypothesize that the reason may be due to lower cell-cell adhesion. ILC spans the spectrum of indolent (benign) to high risk (bad actor) disease. Thus, biomarkers like CTCs may allow us to identify ILC patients who are at higher risk of late recurrence and make appropriate therapeutic decisions at earlier point in time. Studies like Endocrine Optimization Pilot in I-SPY 2 are ongoing to further investigate the use of biomarkers like CTCs to inform outcomes. Due to the short follow-up period, we will conduct an additional follow-up which should give us 10 years of follow-up, which is likely needed due to the risk for late recurrence in ILC patients. Citation Format: Silver Alkhafaji, Denise Wolf, Mark Magbanua, Laura Van 't Veer, John Park, Laura J. Esserman, Rita A. Mukhtar. Differences in levels of circulating tumor cells (CTC) and disseminated tumor cells (DTC) in early-stage lobular versus ductal breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-02.

The relationship between the number of circulating tumor cells and the prognosis in patients with esophageal squamous cell carcinoma.

Esophageal cancer (EC) is one of the most common malignancies worldwide, with high morbidity and mortality rates. Circulating tumor cell (CTC) detection has become a novel approach in clinical study of EC. In this study, the relationship between CTCs/c-Kit expression of CTCs and the prognosis of EC patients was analyzed in EC. A total of 43 EC patients with R0 resection were recruited for this study. The CanPatrol method was used to detect the CTC number in the peripheral blood of patients before and after operations, and the epithelial/interstitial type was classified. Multiple RNA in situ hybridization (RNA-ISH) was used to observethe c-Kit expression of CTCs. Post-operation follow-up occurred over 3 years. Logistic regression or the Cox proportional risk regression model was applied to analyze the relationship between CTC number, CTCs and disease characteristics, pathological stages and prognosis of patients with EC, and changes in CTCs before and after operations. c-Kit expression in different CTCs and the relationship between c-Kit expression and prognosis were also studied. The detection rate of CTCswas 81% (35/43). The detection rates of epithelial-, mixed- and stromal-type CTCs were 53%, 63%, and 33%, respectively. The 3-year overall survival rate was 67%. A CTC level of >2 indicated an increased risk of recurrence, metastasis, and death (P=0.018, 0.002, respectively). Following the operations, the total number of CTCs decreased in 29 cases. Of these, 6 cases were unchanged, and 8 cases demonstrated elevated CTCs. There was a significant difference in the positive rate of mixed-type CTCs before and after the operations. The rate of c-Kit expression in CTCs of EC patients was 46% pre-operation. No statistically significant correlations were found between c-Kit expression and postoperative recurrence/metastasis/survival of EC patients. Preoperative CTC numbers, especially interstitial CTCs, were used as an auxiliary index in the prognosis of EC patients. The mRNA expression of c-Kit was detected in CTCs preoperatively in patients with EC, but no significant correlation between the c-Kit expression and the prognosis of EC patients was found.

Association of Circulating Tumor Cells with Inflammatory and Biomarkers in the Blood of Patients with Metastatic Castration-Resistant Prostate Cancer.

The identification of specific biomarkers that recognize the functional drivers of heterogeneity in prostate cancer (PCa) and personalized treatment remain challenging in systemic medicine. Liquid biopsy allows for the detection and analysis of personalized predictive biomarkers in single blood samples and specifies the current stage of cancer. The aim of our preliminary study was to investigate the association between an elevated circulating tumor cell (CTC) count and the levels of inflammatory factors (IL-6 and IL-8) and biomarkers (DKK-1, PSA, sHER2, and CD44) in patients with metastasized castration-resistant PCa (mCPRC) under chemotherapy and those with localized PCa. Such an association could be used as a component of cancer progression monitoring. We compared the sensitivity and specificity of two CTC isolation platforms. Twenty-eight patients (12 mCRPC and 16 localized PCa patients) were enrolled. Over the study period, the CTC detection rates were 84% with CellCollector® and 73.5% with CellSearch® System in mCPRC patients. The CTC counts determined by the CellSearch® System (CTC_CS) were correlated significantly with the DKK-1, sHER-2, and PSA concentrations in mCRPC patients. The CTC counts captured by CellCollector® demonstrated no significant association with the concentrations of the tested blood-based biomarkers. The CTC_CS count (AUC = 0.9 (95% CI: 0.72–1.0)) and the PSA level (AUC = 0.95 (95% CI: 0.83–1.0)) presented approximately the same sensitivity and specificity for the overall survival of mCRPC patients. For better personalized characterization, further research on CTC phenotyping and their interactions with tumor-associated blood-released factors is needed.

Circulating tumor cell number and endocrine therapy index in ER positive metastatic breast cancer patients

Circulating tumor cells (CTC) are prognostic in metastatic breast cancer (MBC). The CTC-endocrine therapy index (CTC-ETI), consisting of CTC-ER (estrogen receptor), BCL2, human epidermal growth factor receptor (HER2), and Ki67 expression, might predict resistance to endocrine therapy (ET) in patients with ER-positive MBC. One hundred twenty-one patients with ER-positive/HER2-negative MBC initiating a new ET after ≥1 lines of ET were enrolled in a prospective, multi-institutional clinical trial. CTC-ETI and clinical/imaging follow-up were performed at baseline and serial time points. Progression-free survival (PFS) and rapid progression (RP; determined at the 3-month time point) were primary endpoints. Associations with clinical outcomes used logrank and Fisher’s exact tests. At baseline, 36% (38/107) of patients had ≥5 CTC/7.5 ml whole blood (WB). Patients with ≥5 vs. <5 CTC/7.5 ml WB had significantly worse PFS (median 3.3 vs. 5.9 months, P = 0.03). Elevated CTC at 1 month was associated with even worse PFS (1.9 vs. 5.0 months from the 1-month sample, P < 0.001). Low, intermediate, and high CTC-ETI were observed in 71 (66%), 8 (8%), and 28 (26%) patients, with median PFS of 6.9, 8.5, and 2.8 months, respectively (P = 0.008). Patients with high vs. low CTC and CTC-ETI more frequently experienced RP (CTC: 66% vs. 41%; P = 0.03; CTC-ETI: 79% vs. 40%; P = 0.002). In conclusion, CTC enumeration and the CTC-ETI assay are prognostic at baseline and follow-up in patients with ER-positive/HER2-negative MBC starting new ET. CTC at first follow-up might identify a group of patients with ER-positive MBC that could forego ET, but CTC-ETI did not contribute further.

Isolation and Enumeration of Circulating Tumor Cells (CTC) as Prognostic and Predictive Biomarkers in Post-Operative m-CRC

Circulating Tumor Cells (CTCs) presents non-invasive, repeatable investigation of patient‘s disease. In metastatic Colorectal Cancer (m-CRC) patients, CTC enumerations have been comprehensively studied in evaluating metastatic disease. CTC analysis has been shifting from enumeration to more sophisticated molecular depiction of tumor cells, which is used for liquid biopsy of the tumor, reflecting cytological and molecular changes in metastatic patients over time. In this study, CTC enumeration in advanced and localized metastatic colorectal cancer, highlights the vital gains as well as the challenges posed by various approaches, and their implications for advancing disease management. Detection of circulating tumor cells (CTC‘s) or circulating free tumor DNA (ctDNA) to conduct chemotherapy and reporting prognosis is extremely important, In view of the detail CTC has the potential to offer multiple samples by way of sequential minimally-invasive liquid biopsies. In fastidious, there is escalating evidence for the efficacy of CTC‘s in the clinical management of metastatic colorectal cancer (CRC). With most studies confirming the association of elevated CTC counts with worse prognosis. CTC‘s were first identified by Ashworth in 1869. CTC research has been vulnerable by the failure to constantly detect these typical cells. While the normal range of WBCs in human blood is 4.5-119/L, there may only be a few CTC‘s. The most widely used CTC enumeration platform, Cell-Search (Veridex LLC, NJ, USA) was approved for clinical use. As treatment options expand for metastatic colorectal cancer (mCRC), a blood marker with a prognostic and predictive role could guide treatment. Investigations were carried out that Circulating Tumor Cells (CTCs) could predict clinical prognosis in patients with mCRC. This pilot study, demonstrates that CTCs can serve as both prognostic and predictive factor for patients with mCRC. The presence of at least three CTCs at baseline and follow-up is a strong independent prognostic factor for inferior PFS and OS. When utilized in combination with imaging studies, CTCs provide additional prognostic information. There are several studies for which CTCs could have efficacy inmetastatic colorectal cancer. The statistics suggests that CTCs may be used as a stratification feature in metastatic disease treatment trials. The current list of validated prognostic factors is short, with only routine status being universally recognized. Further study should prospectively deal with modification of regimens based on unfavorable CTCs early in the course of treatment will result in enhancement in PFS or OS. As treatment has become more effective for metastatic disease, decision making has become more complicated. Five classes of drugs are on hand for treatment. The most common initial chemotherapy is a fluoropyrimidine with oxaliplatin or irinotecan. CTC levels drawn at 3 to 5 weeks and 6 to 12 weeks, before PET imaging, may lead to prospective regimen choices and standby patients from unnecessary drug toxicity by suggesting that an early change in treatment is defensible.

Circulating tumor cells after neoadjuvant chemotherapy are related with recurrence in esophageal squamous cell carcinoma.

Circulating tumor cells (CTCs) are known to be a systemic process of malignant progression of cancer cells and there is a possibility that analysis for CTCs as a liquid biopsy become predictive or prognostic tools for cancer patients. In the present study with the novel CTCs detection system (Celsee system®), we performed quantitative and qualitative analysis of CTCs in patients with esophageal squamous cell carcinoma (ESCC) receiving neoadjuvant chemotherapy (NAC) with 5FU + CDDP regimen. CTCs are defined as having both DAPI positive and CD45 negative. Vimentin-positive CTCs were defined as mesenchymal-type CTCs (M-CTCs), while epithelial-type CTCs (E-CTCs) were only positive for pan-cytokeratin. At the baseline, there are detectable amounts of CTCs in all patients (n = 30) at all stages, and there were no significant differences of total CTCs, E-CTCs, or M-CTCs numbers between stages. Of importance, among total CTCs, M-CTCs are more dominant than E-CTCs in number. Also, there was no significant change of detectable amounts and phenotype of CTCs before and after NAC (n = 24). Of note, early recurrent group indicated that there was an elevated total CTCs number before NAC and an increased M-CTCs after NAC in comparison to those in non-recurrent group. Quantitative and qualitative analysis of CTCs may provide useful complementary predictive and prognostic information in ESCC.

Pilot study of anti-prostate-specific membrane antigen (PSMA) antibody J591 for men with metastatic castration-resistant prostate cancer (mCRPC) and unfavorable circulating tumor cell (CTC) count.

120 Background: Elevated CTC counts are associated with a poor prognosis in men with mCRPC. PSMA targeted radionuclide therapy has been associated with decline in CTC count, but it remains unclear whether this effect results from radionuclide-induced cytotoxicity. J591 was engineered to have antibody-dependent cytotoxicity. A subset of patients was observed to have CTC count decline following imaging with 111In-J591, so a prospective study was launched to test the hypothesis that “naked” J591 leads to CTC count decline. Methods: In a Simon 2-stage dose de-escalation study, men with progressive mCRPC and unfavorable CTC count (CellSearch &gt; 4) received a single dose of J591. Initial dose cohort 300 mg with de-escalation to 20 mg. CTC count was re-assessed 4, 8, and 12 weeks following therapy along with PSA and standard imaging. An optional PSMA PET was included prior to treatment. The primary endpoint was proportion of subjects with conversion to favorable CTC count ( &lt; 5 CTCs/7.5 mL blood) and/or &gt; 30% decline from baseline within 12 weeks post-treatment. Results: 10 men were enrolled, 9 of whom were evaluable (1 died of progressive mCRPC prior to post-treatment CTC count). Median age was 71.5 years (range 60-81), 78% had prior chemo, ECOG PS 1 in 45% and 2 in 55%. 7 of 9 (78%) evaluable subjects were Halabi CALGB prognostic poor risk category and 2 (22%) intermediate. 6 of 9 had pre-treatment PSMA PET/CT (three 89Zr-J591 and three 68Ga-PSMA11). Though not required, all scans showed &gt; 1 lesion with SUVmax &gt; liver SUV (range 9.12-70.15). 2 of 6 in the 300 mg cohort had CTC count decline; 1 of 6 converted to favorable count (9 to 0 with decrease of 35 to 12 in other). 3 were treated with 20 mg; 1 had CTC count decline of 316 to 112, but 0 converted to favorable count. Across both cohorts, 3 of 9 had a CTC count decline at any point in time, ranging from 65-100% decline. With the pre-specified 2-stage design, enrollment was halted for futility based upon the primary endpoint of 12-week CTC count. PSA values post-treatment increased in 8 (89%) patients and remained unchanged in 1 (11%) patient. Conclusions: Single-agent anti-PSMA antibody J591 may lead to decline in CTC count, though the study did not meet its primary endpoint. A combination or maintenance approach might be preferable and is worthy of exploration.

Abstract PS2-19: Circulating tumor cell (CTC) enumeration in a phase II trial of pazopanib in addition to endocrine therapy in patients with hormone resistant advanced breast cancer (ABC)

Abstract Background: CTCs are detectable in approximately 50% of pts with metastatic breast cancer, and elevated CTC levels are an independent negative prognostic factor. CTCs have been shown to play a valuable role in predicting outcomes in pts with ABC treated with chemotherapy or endocrine therapy. This phase II trial was conducted to evaluate the clinical benefit (CB) of pazopanib, a VEGF receptor tyrosine kinase inhibitor (TKI) combined with nonsteroidal aromatase inhibitors (NSAIs) in pts with ABC resistant to NSAIs. As an exploratory analysis, we hypothesized that CTC levels might be correlated with response to pazopanib plus endocrine therapy. Methods: Eligibility included postmenopausal women with hormone receptor positive (HR+) ABC and progressive disease after at least one month of NSAIs. Treatment was pazopanib 800 mg/day plus either letrozole or anastrozole. The primary endpoint was clinical benefit rate at 12 weeks (CBR12, wks). Secondary endpoints included progression free survival (PFS), safety, and the impact of pazopanib and NSAI on CTCs. A CBR of 20% was considered a clinically meaningful comparison to the expected CBR of &amp;lt;5% with continued NSAIs after PD. Blood samples were collected for CTC analysis at baseline, then every 4 wks while on treatment. The presence of CTCs was assessed using the FDA-cleared CellSearch System; CTCs were defined as EPCAM-positive, CD45-negative nucleated cells. Samples with 5 CTCs per 7.5 mLs of blood were considered CTC-positive. Results: 32 pts were enrolled; 28 are evaluable for study endpoints. The median age was 58 years (range: 41-77). Pts were heavily pre-treated, with a median of 2 prior hormone therapies (range 1-6) and 1 prior chemotherapy (range 0-8). 8 pts (28.6%) stopped treatment due to adverse events, and 6 pts progressed before wk 12. CBR12 was 46.4% (12 SD, 1 PR), and CBR24 was 25% (5 SD, 2 PR). Safety has been presented (ASCO 2020). Median PFS was 20 wks, and median PFS for pts with CBR12 was 24 wks. Five (22%) of 23 pts with CTC data were CTC-positive (5 CTC/7.5 mL). There was no significant association between CBR12 and CTC status at baseline, or between CBR12 and change in CTC status (baseline to 4 wks after initiation of treatment). 7 pts had non-CBR12 and CTC data; 4 pts (n=4/7; 57%) were CTC positive at either baseline, week 4 or both. 11 pts had CBR12 and CTC data; all patients were CTC negative at baseline and remained negative at 4 wks. 7 pts had PFS &amp;gt;6 months (24, 32, 36, 36, 48, 184 and 274 wks), and 6 had CTC data: all were CTC-negative at baseline and 4 wks, then remained negative at 12 wks. Based on baseline values only, CTC-positive pts had a shorter median PFS compared to CTC-negative pts, but the difference was not statistically significant (11 wks vs 14 wks, p=0.18). Persistently CTC negative patients had significantly longer PFS compared to patients who were CTC positive at any time during study treatment (36 wks vs 11 wks, p=0.01). Conclusions: The addition of pazopanib to NSAIs resulted in a CBR12 of 46.4%, and a CBR24 of 25% in pts with heavily pre-treated ABC resistant to NSAIs. These results support clinical efficacy of antiangiogenic TKI in HR+ ABC and suggest benefit in hormone resistant disease. Consistently negative CTCs correlated with response and response duration. PFS was significantly longer in patients who were consistently CTC negative, further defining the prognostic role of CTCs in HR+ ABC. (NCT01466972) Citation Format: Ozge Gumusay, Mark Jesus M. Magbanua, Melanie C. Majure, Travis Deal, Jonathan R. Renslo, Chiara A. Wabl, Michelle E. Melisko, A. Jo Chien, Andrei Goga, Mark M. Moasser, John W. Park, Jimmy Hwang, Hope S. Rugo. Circulating tumor cell (CTC) enumeration in a phase II trial of pazopanib in addition to endocrine therapy in patients with hormone resistant advanced breast cancer (ABC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-19.

Analysis of a Real-World Cohort of Metastatic Breast Cancer Patients Shows Circulating Tumor Cell Clusters (CTC-clusters) as Predictors of Patient Outcomes.

Circulating tumor cell (CTC) enumeration has emerged as a powerful biomarker for the assessment of prognosis and the response to treatment in metastatic breast cancer (MBC). Moreover, clinical evidences show that CTC-cluster counts add prognostic information to CTC enumeration, however, their significance is not well understood, and more clinical evidences are needed. We aim to evaluate the prognostic value of longitudinally collected single CTCs and CTC-clusters in a heterogeneous real-world cohort of 54 MBC patients. Blood samples were longitudinally collected at baseline and follow up. CTC and CTC-cluster enumeration was performed using the CellSearch® system. Associations with progression-free survival (PFS) and overall survival (OS) were evaluated using Cox proportional hazards modelling. Elevated CTC counts and CTC-clusters at baseline were significantly associated with a shorter survival time. In joint analysis, patients with high CTC counts and CTC-cluster at baseline were at a higher risk of progression and death, and longitudinal analysis showed that patients with CTC-clusters had significantly shorter survival compared to patients without clusters. Moreover, patients with CTC-cluster of a larger size were at a higher risk of death. A longitudinal analysis of a real-world cohort of MBC patients indicates that CTC-clusters analysis provides additional prognostic value to single CTC enumeration, and that CTC-cluster size correlates with patient outcome.

Circulating Tumor Cells and Metabolic Parameters in NSCLC Patients Treated with Checkpoint Inhibitors.

Circulating tumor cells (CTC) count and characterization have been associated with poor prognosis in recent studies. Our aim was to examine CTC count and its association with metabolic parameters and clinical outcomes in non-small cell lung carcinoma (NSCLC) patients treated with immune checkpoint inhibitors (ICI). For this prospective study, data from 35 patients (23 males, 12 females) were collected and analyzed. All patients underwent an 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) scan and CTC detection through Isolation by Size of Tumor/Trophoblastic Cells (ISET) from peripheral blood samples obtained at baseline and 8 weeks after ICI initiation. Association of CTC count with clinical and metabolic characteristics was studied. Progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan–Meier method and the log-rank test. Median follow-up was 13.2 months (range of 4.9–21.6). CTC were identified in 16 out of 35 patients (45.7%) at baseline and 10 out of 24 patients at 8 weeks (41.7%). Mean CTC numbers before and after 8 weeks were 15 ± 28 and 11 ± 19, respectively. Prior to ICI, the mean CTC number was significantly higher in treatment-naïve patients (34 ± 39 vs. 9 ± 21, p = 0.004). CTC count variation (ΔCTC) was significantly associated with tumor metabolic response set by European Organization for Research and Treatment of Cancer (EORTC) criteria (p = 0.033). At the first restaging, patients with a high tumor burden, that is, metabolic tumor volume (MTV) and total lesion glycolysis (TLG), had a higher CTC count (p = 0.009). The combination of mean CTC and median MTV at 8 weeks was associated with PFS (p < 0.001) and OS (p = 0.024). Multivariate analysis identified CTC count at 8 weeks as an independent predictor for PFS and OS, whereas ΔMTV and maximum standardized uptake value variation (ΔSUVmax) was predictive for PFS and OS, respectively. Our study confirmed that CTC number is modulated by previous treatments and correlates with metabolic response during ICI. Moreover, elevated CTC count, along with metabolic parameters, were found to be prognostic factors for PFS and OS.

Abstract P6-10-23: Presence of circulating tumor cell clusters and elevated platelet counts interactively predict disease progression in advanced-stage breast cancer patients

Abstract Background: Circulating tumor cells (CTCs) and their clusters (CTC-clusters) are promising prognostic markers in breast cancer. Recently studies showed that platelets interact with tumor cells in the circulation and promote metastasis. However, no study has been reported to explore the effects of interactions between CTCs/CTC-clusters and platelets on the prognosis of breast cancer at the population level. Methods: A total of 146 female advanced-stage breast cancer patients were included in this study. CTCs and CTC-clusters were enumerated using the CellSearch system. Platelet count was obtained from laboratory tests. The individual and joint effects of CTCs/CTC-clusters and platelets on progression free survival (PFS) were evaluated using the Cox proportional hazards model. Results: Elevated CTCs (≥ 5 CTCs/7.5 mL), presence of CTC-clusters, or high platelet counts (≥ 400 × 109/L) were individually associated with PFS (hazard ratio [HR] 2.69, 2.71, and 1.79, respectively). We further stratified patients into three risk groups according to the status of CTCs/CTC-clusters in combination with the level of platelet counts, including: 1) low-risk group: for CTC-related analyses, low CTCs (&amp;lt; 5 CTCs/7.5 mL) and platelet counts (&amp;lt; 400 × 109/L); for CTC-cluster-related analyses, absence of CTC-cluster and low platelet counts; 2) medium-risk group: for CTC-related analyses, either elevated CTCs (≥ 5 CTCs/7.5 mL) or high platelet counts (≥ 400 × 109/L), but not both; for CTC-cluster-related analyses, either presence of CTC-clusters or high platelet counts, but not both; 3) high-risk group: for CTC-related analyses, both elevated CTCs and high platelet counts; for CTC-cluster-related analyses, presence of CTC-clusters and high platelet counts. A significantly increased risk for disease progression was observed in high-risk patients, especially in patients with both CTC-clusters and high platelet counts (adjusted HR 16.06, P = 0.0003, log-rank P&amp;lt; 0.0001). Furthermore, we identified a multiplicative interaction between CTC-clusters and platelets on PFS (P for interaction = 0.0483), and the patients with both CTC-clusters and high platelet counts had the shortest time of development of new metastatic sites. Conclusions: Our results indicated the effects of interactions between the presence of CTC-clusters and high platelet counts on tumor progression and dissemination in breast cancer. Future studies are warranted to validate our findings and explore their biological mechanisms and clinical relevance. Citation Format: Chun Wang, Zhong Ye, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, AnaMaria Lopez, Frederick Fellin, Saveri Bhattacharya, Rebecca Jaslow, Kaelan Yao, Darayus Toorkey, Neha Pancholy, Ronald Myers, Hushan Yang. Presence of circulating tumor cell clusters and elevated platelet counts interactively predict disease progression in advanced-stage breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-23.

First-Line Doublet Chemotherapy for Metastatic Triple-Negative Breast Cancer: Circulating Tumor Cell Analysis of the tnAcity Trial.

PurposeCirculating tumor cells (CTCs) are prognostic biomarkers in metastatic breast cancer, but their role in predicting treatment outcomes in metastatic triple-negative breast cancer (mTNBC) is less clear. The tnAcity trial demonstrated a significant progression-free survival (PFS) benefit with nab-paclitaxel (nab-P)/carboplatin (C) over nab-P/gemcitabine (G) or G/C in patients with mTNBC. We assessed the correlation between CTC dynamics and clinical benefit in all patients and by treatment arm.MethodsCTC enumeration, performed using CELLSEARCH technology (Menarini Silicon Biosystems, Huntingdon Valley, PA, USA), was a prespecified exploratory endpoint in the tnAcity trial. Patients with TNBC were categorized based on pre- and post-treatment CTC levels: Group 1 (+ + +; elevated CTCs at baseline and postbaseline), Group 2 (+ ± ±; CTCs elevated at baseline and cleared postbaseline [cycle 3 and/or cycle 5]), or Group 3 (−; no CTCs detected at baseline). The baseline cutoff was ≥1 CTC/7.5 mL for the main analysis; cutoffs of ≥2 and ≥5 CTCs were used for supporting analyses.ResultsThe main analysis included 126 patients (Group 1, n = 24; Group 2, n = 54; and Group 3, n = 48). The median PFS was longer in Group 2 vs Group 1 (8.5 vs 4.7 months; HR, 0.30 [95% CI, 0.17–0.54]). These results were supported by the ≥2- and ≥5-CTC cutoff analyses. The median overall survival rates were 17.8, 16.0, and 9.8 months in Groups 2, 3, and 1, respectively. The overall response rates were 79.6%, 43.8%, and 29.2%, respectively. A numerically higher percentage of patients had CTC clearance during nab-P/C treatment vs nab-P/G or G/C.ConclusionEfficacy outcomes trended positively with chemotherapy-induced elimination of CTCs, suggesting that CTC clearance may predict the chemosensitivity of mTNBC tumors.Trial registrationEudraCT Number: 2013-000113-20; ClinicalTrials.gov number: NCT01881230.